The detection and quantification of biological material, such as cells, proteins, e.g. cytokines and antibodies, drugs, nucleic acid, e.g. DNA and RNA, etc., using fluorescent labeling or enzyme-processed chromogens is widely applied in different biological assay systems.
The fluorescent dye is particularly useful for assays like immuno histochemistry (IHC), flow cytometry, fluorescent in situ hybridization (FISH) and similar systems. Fluorescent dyes, or fluorochromes, are fluorescent when appropriately excited under normal conditions of use chromogens have different characteristics than fluorochromes and form colored precipitates when processed by an enzyme. Under normal conditions of use chromogens form intensely colored precipitates when viewed using ordinary lighting conditions. Chromogens are frequently used in IHC, and similar enzyme-linked immuno assays.
Most enzyme-linked assays are based on one of the two enzymes alkaline phosphatase (AP), a hydrolase (Self C H, J Imm Methods 76:389-393, 1985) or horseradish peroxidase (HRP), an oxidoreductase (Bystryak, Mekler, Anal Biochem 202:390-393, 1992). Alkaline phosphatase activity is in general expressed through hydrolysis of a reporter substrate and its subsequent reaction with a diazonium salt to generate a detectable signal. HRP oxidizes a reporter substrate and this reporter substrate will then through polymerization or covalent attachment generate a detectable signal.
Cyanine dyes and related polymethine dyes are well known in the literature (Tyutyulkov, N., et al., Polymethine dyes: Structure and properties. 1st ed. 1991: St. Kliment Ohridski University press. 249). Furthermore, modifications of the dyes to provide desired solubility, reactivity and spectroscopic properties have also been suggested (Tyutyulkov, N., et al., Polymethine dyes: Structure and properties. 1st ed. 1991: St. Kliment Ohridski University press. 249).
Cyanine dyes as substrate for enzymes has been described previously, however only in systems where the dyes are coupled to a molecule that is known to be a substrate for an enzyme (in this case horse radish peroxidase (HRP)). Chao et al. (Cytometry 23:48-53, 1996) describes a fluorescent horseradish peroxidase substrate Cy3.29-tyramide and its application in an enzyme-based signal amplification system (catalyzed reporter deposition, CARD).
EP 747 448 describes fluorescent monomethine rigidized dye compounds emitting near UV and blue (300-500 nm) region of the spectrum.
U.S. Pat. No. 6,686,145 describes fluorescent rigidized dye compounds capable of producing fluorescence in the green to orange region of the spectrum.
U.S. Pat. No. 5,268,486 describes cyanine and polymethine dyes developed with substituent groups which are covalently reactive with sulfhydryl groups, amine groups, and hydroxyl groups on proteins and other materials for purposes of fluorescence and phosphorescence detection of those materials.
U.S. Pat. No. 5,569,587 describes to labeling of proteins, DNA, drugs, blood cells, etc. with luminescent polymethine cyanine and polymethine dyes at an amine or hydroxyl site on those materials.
Complex analysis of biological material demands parallel analysis of several biological molecules to obtain the desired information. Today's assay systems are most often limited by the number of colors available to perform such analysis of several markers in different colors on one tissue section.
It is thus highly desirable to develop means and methods for more colors to be available in biological assays to allow for e.g. parallel analysis of biological markers and molecules, and to allow manipulation of the color of the dye to suit specific needs of the user, such as specific color needs or choice of fluorescence or visual detection systems, in an easy and simple way. In this respect the present invention addresses this need and interest.